Metal ion-mediated polymer superquenching for highly sensitive detection of kinase and phosphatase activities

doi:10.1073/pnas.0406832101

Metal ion-mediated polymer superquenching for highly sensitive detection of kinase and phosphatase activities

QTL Biosystems, 2778 Agua Fria Street, Santa Fe, NM 87507

Communicated by Alan J. Heeger, University of California, Santa Barbara, CA, September 15, 2004 (received for review April 25, 2004)

Abstract

An assay technology for high-throughput screening of kinase and phosphatase activities is introduced. The format is based upon superquenching of fluorescent-conjugated polymers by dye-labeled kinase/phosphatase peptide substrates. The sensor platform is composed of highly fluorescent-conjugated polyelectrolytes colocated with the phosphate coordinating metal ion gallium on microspheres. Phosphorylated peptide substrates containing a quencher bind specifically to the metal ions by means of phosphate groups, resulting in quench of polymer fluorescence. The modulation of fluorescence signal is proportional to kinase or phosphatase activity and is monitored as a turn-off or turn-on signal, respectively. The assay is homogeneous and simple and can be run either as an endpoint measurement or in a kinetic mode. The assay meets the sensitivity required for high-throughput screening of kinase or phosphatase inhibitors and is a valuable tool for drug discovery. A modified version of the assay allows for the detection of protein phosphorylation.

Footnotes

↵ * To whom correspondence should be addressed. E-mail: whitten{at}qtlbio.com.
Author contributions: F.R., K.A., D.M., and D.W. designed research; F.R., W.X., S.W., X.S., and C.S. performed research; F.R., W.X., and S.W. analyzed data; and F.R., K.A., D.M., and D.W. wrote the paper.
Abbreviations: EC₅₀, enzyme concentration at which 50% substrate is converted; FRET, fluorescence resonance energy transfer; IC₅₀, inhibitor concentration that reduces the enzyme activity by 50%; MBP, myelin basic protein; PKA, protein kinase A; PPE, poly (p-phenylenethynylene); PTP-1B, protein tyrosine phosphatase 1B; QTL, quencher-tetherligand; RFU, relative fluorescence units; mU, milliunit.
↵ † Solid GaCl₃ was dissolved in buffer at pH 5.5; it is reported (30–32) that aqueous solutions of Ga(III) are subject to hydrolysis and may exist as various polymeric gallium-oxo-species. It is therefore not possible to specify the exact gallium species that associates with the sensor.